Deciphering Opioid Peptide Binding Modes at Atypical Chemokine Receptor 3

ACKR3 is a class A G protein-coupled receptor that is considered as an atypical chemokine receptor. It does not activate G proteins but efficiently recruits β-arrestin and mediates ligand internalization and was thus proposed as a scavenger receptor. Besides chemokines, ACKR3 internalizes a variety of endogenous opioid peptides, including adrenorphin and dynorphin A. By reducing their availability to the classical opioid receptors, ACKR3 is proposed to participate in the endogenous pain management system, suggesting it as a new potential target for a new class of analgesics. Available structural data for ACKR3 are focused on the binding of chemokines (e.g., CXCL12), but how opioid peptides bind at ACKR3 remains enigmatic. Here, we structurally modeled opioid peptide binding at ACKR3 with a focus on adrenorphin, its ACKR3 selective variant LIH383, and dynorphin A. By combining molecular dynamics simulations with pharmacophore analysis, we analyze the opioid peptides' binding modes and compare them with binding to classical opioid receptors (MOR, KOR, and DOR). We apply our model to rationally explain previously reported structure-activity relationships for adrenorphin derivatives, which also supports the model's validation. Moreover, we include <i>in vitro</i> ACKR3 mutational experiments on both the receptor and LIH383 to further strengthen our structural model. Taken together, we systematically combine <i>in silico</i> observations and <i>in vitro</i> readouts to contribute to the understanding of ACKR3's ligand binding profile and set the basis for further ACKR3 ligand development.